Analysis of bacterial transcriptome and epitranscriptome using nanopore direct RNA sequencing.

Bacterial gene expression is a complex process involving extensive regulatory mechanisms. Along with growing interests in this field, Nanopore Direct RNA Sequencing (DRS) provides a promising platform for rapid and comprehensive characterization of bacterial RNA biology. However, the DRS of bacterial RNA is currently deficient in the yield of mRNA-mapping reads and has yet to be exploited for transcriptome-wide RNA modification mapping. Here, we showed that pre-processing of bacterial total RNA (size selection followed by ribosomal RNA depletion and polyadenylation) guaranteed high throughputs of sequencing data and considerably increased the amount of mRNA reads. This way, complex transcriptome architectures were reconstructed for Escherichia coli and Staphylococcus aureus and extended the boundaries of 225 known E. coli operons and 89 defined S. aureus operons. Utilizing unmodified in vitro-transcribed (IVT) RNA libraries as a negative control, several Nanopore-based computational tools globally detected putative modification sites in the E. coli and S. aureus transcriptomes. Combined with Next-Generation Sequencing-based N6-methyladenosine (m6A) detection methods, 75 high-confidence m6A candidates were identified in the E. coli protein-coding transcripts, while none were detected in S. aureus. Altogether, we demonstrated the potential of Nanopore DRS in systematic and convenient transcriptome and epitranscriptome analysis.

Primerdiffer: a python command-line module for large-scale primer design in haplotype genotyping.

MOTIVATION: Primer design is a routine practice for modern molecular biology labs. Bioinformatics tools like primer3 and primer-blast have standardized the primer design for a specific region. However, large-scale primer design, especially for genome-wide screening, is still a labor-intensive job for most wet-lab researchers using these pipelines. RESULTS: Here, we present the primerdiffer pipeline, which can be used to batch design primers that differentiate haplotypes on a large scale with precise false priming checking. This command-line interface (CLI) pipeline includes greedy primer search, local and global in silico PCR-based false priming checking, and automated best primer selection. The local CLI application provides flexibility to design primers with the user's own genome sequences and specific parameters. Some species-specific primers designed to genotype the hybrid introgression strains from Caenorhabditis briggsae and Caenorhabditis nigoni have been validated using single-worm PCR. This pipeline provides the first CLI-based large-scale primer design tool to differentiate haplotypes in any targeted region. AVAILABILITY AND IMPLEMENTATION: The open-source python modules are available at github (https://github.com/runsheng/primerdiffer, https://github.com/runsheng/primervcf) and Python package index (https://pypi.org/project/primerdiffer/, https://pypi.org/project/primervcf/).

Nanopore Current Events Magnifier (nanoCEM): a novel tool for visualizing current events at modification sites of nanopore sequencing.

Nanopore sequencing technologies have enabled the direct detection of base modifications in DNA or RNA molecules. Despite these advancements, the tools for visualizing electrical current, essential for analyzing base modifications, are often lacking in clarity and compatibility with diverse nanopore pipelines. Here, we present Nanopore Current Events Magnifier (nanoCEM, https://github.com/lrslab/nanoCEM), a Python command-line tool designed to facilitate the identification of DNA/RNA modification sites through enhanced visualization and statistical analysis. Compatible with the four preprocessing methods including 'f5c resquiggle', 'f5c eventalign', 'Tombo' and 'move table', nanoCEM is applicable to RNA and DNA analysis across multiple flow cell types. By utilizing rescaling techniques and calculating various statistical features, nanoCEM provides more accurate and comparable visualization of current events, allowing researchers to effectively observe differences between samples and showcase the modified sites.

Giraffe: A tool for comprehensive processing and visualization of multiple long-read sequencing data.

Third-generation sequencing techniques have become increasingly popular due to their capacity to produce long, high-quality reads. Effective comparative analysis across various samples and sequencing platforms is essential for understanding biological mechanisms and establishing benchmark baselines. However, existing tools for long-read sequencing predominantly focus on quality control (QC) and processing for individual samples, complicating the comparison of multiple datasets. The lack of comprehensive tools for data comparison and visualization presents challenges for researchers with limited bioinformatics experience. To address this gap, we present Giraffe (https://github.com/lrslab/Giraffe_View), a Python3-based command-line tool designed for comparative analysis and visualization across diverse samples and platforms. Giraffe facilitates the assessment of read quality, sequencing bias, and genomic regional methylation proportions for both DNA and direct RNA sequencing reads. Its effectiveness has been demonstrated in various scenarios, including comparisons of sequencing methods (whole genome amplification vs. shotgun), sequencing platforms (Oxford Nanopore Technology, ONT vs. Pacific Biosciences, PacBio), tissues (kidney marrow with and without blood), and biological replicates (kidney marrows).

Coalbed Methane Enrichment Characteristics and Exploration Target Selection in the Zhuozishan Coalfield of the Western Ordos Basin, China.

The Zhuozishan coalfield at the western margin of the Ordos Basin is one of the main coal-mining areas in China, and recent explorations have revealed the great potential for coalbed methane (CBM) resources in its Carboniferous and Permian strata. In this paper, the controlling factors of CBM enrichment of the major coals are studied in this coalfield and the CBM resources are estimated based on the analysis of the coal petrology and compilation of literature data on the gas content. The result of the coal petrology analysis of 10 samples shows that the vitrinite content of No. 16 coal (71.9-77.3%) is higher than that of No. 9 coal (59.1-65.1%), and the inertinite content of No. 16 coal (18.9-23.5%) is lower than that of No. 9 coal (30.1-34.9%). The R o,max value of No. 16 coal (1.18-1.35%) is higher than that of No. 9 coal (1.04-1.13%), and both coals are of medium rank. Due to greater thickness, deeper burial depth, and better coal petrology characteristics, the No. 16 coal seam of the Taiyuan Formation is selected as the major coal seam for CBM resource estimation, which has a thickness of 1-6 m and a present-day burial depth of 200-1100 m. The gas content of this coal seam varies mostly between 4 and 10 m3/t. Positive correlation between the coal seam thickness as well as present-day burial depth and the gas content suggests that the thick and deeply buried coal seams are favorable for CBM preservation. The ash yield shows an insignificant negative correlation with the gas content, indicating that ash yield is not an important factor for CBM enrichment. The syncline hinges located below the thrust zones show higher gas content due to greater burial depths. In contrast, the anticline hinges at shallower depths tend to have lower gas contents. Based on the combined information about sedimentary environments, structural patterns, and hydrogeology, two CBM accumulation models are put forward in the study area that include syncline-hydraulic plugging below thrust nappe and fault-confined aquifer plugging. The volumetric method is used to estimate the CBM resources, and results indicate that the CBM resource in the whole coalfield is 428.78 × 108 m3, and the total resource abundance is 0.74 × 108 m3/km2. Two favorable areas for the CBM exploration are optimized based on the resource amount and resource abundance. One of the favorable areas is the Kabuqi area in the northern part of the coalfield, and another is the Baiyunwusu area in the central part of the coalfield. These two areas will be the CBM priority exploration areas at the western margin of the Ordos Basin.

High-pressure destruction kinetics of Clostridium sporogenes spores in ground beef at elevated temperatures.

High pressure (HP) is an alternative technique for thermal sterilization of foods with minimum quality loss. HP destruction kinetics of bacterial spores is essential to establishing sterilization process, but knowledge in this field is still very limited. In this study, destruction kinetics was investigated using Clostridium sporogenes PA 3679 (ATCC7955) spores in extra-lean ground beef (5 g each sealed in a sterile plastic bag). Duplicated samples were subjected to HP treatments at 700, 800 and 900 MPa in a HP system equipped with a Polyoxymethylene insulator to maintain constant temperatures at 80, 90 and 100 degrees C during pressure-holding time. The kinetic parameters of the spores (D- and Z-values) were evaluated at these pressures and temperatures. For the pressure from 700 to 900 MPa, D-values ranged from 15.8 to 7.0 and 1.5 to 0.63 min at 80 and 100 degrees C, respectively. The pressure resistance of Z(T)(P) value was 520-563 MPa at 80-100 degrees C. The temperature resistance of Z(P)(T) value was 19.1-19.7 degrees C at 700-900 MPa, much higher than that at atmospheric condition (12.4 degrees C). A regression model was generated which can be used to predict D-value or the death time of a minimum process under given pressure and temperature conditions. HP treatment with elevated temperatures can destroy bacterial spores with a shorter time or lower temperature than conventional thermal processing. This study provides useful information for the achievement of a safe HP sterilization process.